Endolysin polypeptide

ABSTRACT

The invention relates to the field of medicine, specifically to the field of treatment of conditions associated with Staphylococcus infection. The invention relates to a novel endolysin polypeptide specifically targeting a bacterial Staphylococcus cell. The invention further relates to said endolysin polypeptide for medical use, preferably for treating an individual suffering from a condition associated with Staphylococcus infection.

FIELD OF THE INVENTION

The invention relates to the field of medicine, specifically to thefield of treatment of conditions associated with Staphylococcusinfection. The invention relates to a novel endolysin polypeptidespecifically targeting a bacterial Staphylococcus cell. The inventionfurther relates to said endolysin polypeptide for medical use,preferably for treating an individual suffering from a conditionassociated with Staphylococcus infection.

BACKGROUND OF THE INVENTION

Staphylococcus aureus is a major animal pathogen, especially in mammalssuch as humans and cows, which is frequently implicated in severalserious infectious diseases and food poisoning. Its treatment becomesmore and more difficult because of emerging antibiotic resistantstrains. Endolysins from phages infecting Staphylococcus aureus havebeen shown to potentially control these pathogens and can be used fortheir specific detection. In most cases, major obstacles in theapplication of endolysins targeting Staphylococcus species are lowenzyme activity, difficult production in large quantities and/or proteinstability. Accordingly, there is a need for an endolysin polypeptidewith improved characteristics on for example antimicrobial activity,stability and/or reduced immunogenicity.

SUMMARY OF THE INVENTION

The invention relates to an endolysin polypeptide specific forStaphylococcus, said polypeptide comprising:

a polypeptide with an amino acid sequence that has at least 80% sequenceidentity with SEQ ID NO: 2, or

a polypeptide with an amino acid sequence that has at least 80% sequenceidentity with SEQ ID NO: 1 and having a truncation of at least 5, 10,20, 30, or 40 amino acids in the region of amino acids 156 to 199 of SEQID NO: 1.

The invention further relates to an endolysin polypeptide with an aminoacid sequence according to SEQ ID NO: 2.

The invention further relates to a polynucleotide encoding an endolysinpolypeptide according to the invention. The invention further relates toa nucleic acid construct comprising a polynucleotide according to theinvention. The invention further relates to an expression vectorcomprising a nucleic acid construct according to the invention.

The invention further relates to a host cell comprising a polynucleotideaccording to the invention, a nucleic acid construct according to theinvention or an expression construct according to the invention.

The invention further relates to a method for the production of anendolysin polypeptide according to the invention, comprising:

culturing a host cell according to the invention under conditionsconducive to the production of the endolysin polypeptide,

optionally isolating and purifying the endolysin polypeptide from theculture broth, and

optionally freeze-drying the endolysin polypeptide.

The invention further relates to a method for producing an endolysinpolypeptide according to the invention with enhanced activity comprisingdialysis of an endolysin according to the invention, or an endolysinpolypeptide obtainable by a method according to the invention, saiddialysis comprising the steps of:

i) dialysis against a buffer comprising a chelating compound, and

ii) dialysis against a divalent metal ion-containing buffer, preferablya divalent metal ion selected form the group consisting of Co²⁺, Cu²⁺,Mg²⁺, Ca²⁺, Mn²⁺ and Zn²⁺.

The invention further relates to a composition comprising an endolysinpolypeptide according to the invention, or an endolysin polypeptideobtainable by a method according to the invention, or a polynucleotideaccording to the invention, or a nucleic acid construct according to theinvention, or an expression construct according to the invention, or ahost cell according to the invention.

The invention further relates to pharmaceutical composition comprisingan endolysin polypeptide according to the invention, or an endolysinpolypeptide obtainable by a method according to the invention, or apolynucleotide according to the invention, or a nucleic acid constructaccording to the invention, or an expression construct according to theinvention, or a host cell according to the invention, saidpharmaceutical composition further comprising a pharmaceuticallyacceptable excipient.

The invention further relates to a composition according to invention,further comprising an additional active ingredient.

The invention further relates to a composition according to theinvention, for use as a medicament.

The invention further relates to a composition according to theinvention, for use as a medicament in the treatment of a conditionassociated with infection with a Staphylococcus.

The invention further relates to a method of treatment of a conditionassociated with infection with a Staphylococcus, comprisingadministration an endolysin polypeptide according to the invention, oran endolysin polypeptide obtainable by a method according to theinvention, or a polynucleotide according to the invention, or a nucleicacid construct according to the invention, or an expression constructaccording to the invention, or a host cell according to the invention,or a composition according to the invention.

The invention further relates to the use of an endolysin polypeptideaccording to the invention, or an endolysin polypeptide obtainable by amethod according to the invention, or a polynucleotide according to theinvention, or a nucleic acid construct according to the invention, or anexpression construct according to the invention, or a host cellaccording to the invention, or a composition according to the invention,as an antimicrobial, preferably as a food additive or a disinfectant.

The invention further relates to the use of a an endolysin polypeptideaccording to the invention, or an endolysin polypeptide obtainable by amethod according to the invention, or a polynucleotide according to theinvention, or a nucleic acid construct according to the invention, or anexpression construct according to the invention, or a host cellaccording to the invention, or a composition according to the invention,for detecting a Staphylococcus, preferably in a diagnostic application.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, a novel endolysin polypeptide with enhanced specificactivity and reduced immunogenicity was arrived at by the inventors.

Accordingly, in a first aspect, the present invention provides for anendolysin polypeptide specific for Staphylococcus, said polypeptidecomprising:

a polypeptide with an amino acid sequence that has at least 80% sequenceidentity with SEQ ID NO: 2, or

a polypeptide with an amino acid sequence that has at least 80% sequenceidentity with SEQ ID NO: 1 and having a truncation of at least 5, 10,20, 30, or 40 amino acids in the region of amino acids 156 to 199 of SEQID NO: 1.

Said endolysin polypeptide herein referred to as an endolysinpolypeptide according to the invention. Preferably, said endolysinpolypeptide according to the invention has enhanced specific activityand/or reduced immunogenicity than an endolysin polypeptide with theamino acid sequence according to SEQ ID NO: 1. More preferably, saidendolysin polypeptide according to the invention has enhanced specificactivity and reduced immunogenicity than an endolysin polypeptide withthe amino acid sequence according to SEQ ID NO: 1. Preferably, theendolysin polypeptide according to the invention comprises a polypeptidewith an amino acid sequence that has at least 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore preferably 100% sequence identity with SEQ ID NO: 2. Morepreferably, the endolysin polypeptide according to the inventionconsists of a polypeptide with an amino acid sequence that has at least81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more preferably 100% sequence identity withSEQ ID NO: 2.

Preferably, the endolysin polypeptide according to the inventioncomprises a polypeptide with an amino acid sequence that has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or more preferably at least 99% sequenceidentity with SEQ ID NO: 1 and having a truncation of at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 ormore preferably at least 40 amino acids in the region of amino acids 156to 199 of SEQ ID NO: 1. Herein, the term truncation may also be referredto as an internal truncation or as a deletion.

A more preferred endolysin polypeptide according to invention has anamino acid sequence according to SEQ ID NO: 2.

Within the context of the invention, an endolysin polypeptide specificfor Staphylococcus is construed as a polypeptide that has lytic activityfor Staphylococcus, preferably said lytic activity is peptidoglycanhydrolase activity for Staphylococcus peptidoglycan. Preferably, saidStaphylococcus is selected from the group consisting of S. aureus, S.simulans and S. carnosus. Lytic activity can be assessed by any suitablemethod known to the person skilled in the art. Preferably, one of theassays as described in the examples herein is used.

Encompassed within the invention are endolysin polypeptides thatcomprise an endolysin according to the invention or a functional partthereof as a backbone and further comprises a further polypeptide or afunctional part thereof. Said further polypeptide of a functional partthereof may e.g. be, but is not limited to a lytic domain or an amidasedomain. Such further polypeptide or functional part thereof may henative of may be heterologous to the endolysin polypeptide according tothe invention.

An endolysin polypeptide according to the invention may be derived fromone of the sequences herein by substituting, inserting, deleting, oradding one, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 or morenucleotides or amino acids, respectively. An endolysin polypeptideaccording to the invention may be derived from one of the sequences asidentified herein by adding an additional N- or C-terminal amino acidsor chemical moieties to increase stability, solubility and activity.

An endolysin polypeptide according to the invention may have a length ofat least 300, 350, 400 or 450 amino acids, preferably 462 amino acids,and/or at most 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600,500 or 462 amino acids. Preferably, said polypeptide has a length of atleast 400, 410, 420, 430, 440, 450, 460, 462, 470, 480, 490, 500, 510 or520 amino acids and/or at most 1500, 1400, 1300, 1200, 1100, 1000, 900,800, 700, 600 amino acids. An embodiment of the invention encompasses avariant endolysin polypeptide according to the invention. A variantendolysin polypeptide may be any non-naturally occurring form of theendolysin polypeptide according to the invention. An endolysinpolypeptide variant may differ in some engineered way from SEQ DI NO: 2.A variant endolysin polypeptide according to the invention may be madeby site-directed mutagenesis starting from the nucleotide sequence ofSEQ ID NO: 1 or SEQ ID NO: 2. Preferably, a variant endolysinpolypeptide according to the invention contains mutations that do notalter the biological function of the encoded polypeptide; preferably, avariant endolysin polypeptide according to the invention exhibitsStaphylococcus peptidoglycan cell wall-binding and/or a lytic activitywhich is the same or enhanced as compared to the endolysin polypeptidewith the amino acid sequence according to SEQ ID NO: 2.

An endolysin polypeptide according to the invention may comprise a tagfor e.g. ease of purification. Preferably, said tag is selected from,but is not limited to, the group consisting of a FLAG-tag,poly(His)-tag, HA-tag and Myc-tag, more preferably said tag is a 6×His-tag.

In a second aspect, the invention provides for (claim 3) apolynucleotideencoding an endolysin polypeptide according to the first aspect of theinvention. Preferably, such polynucleotide according to the invention iscoding optimized for efficient expression in a specific host. The personskilled in the art knows how to optimize codons and/or codon pairs. Apolynucleotide according to the invention may be an RNA or DNA molecule,preferably a DNA molecule.

The invention further relates to a nucleic acid construct comprising apolynucleotide according to the invention.

The invention further relates to an expression vector comprising anucleic acid construct or a polynucleotide according to the invention.An expression vector according to the invention may be a recombinantexpression vector. Such vector may constitute a plasmid, a cosmid, abacteriophage or a virus, or a part thereof, which is transformed byintroducing a nucleic acid construct or a polynucleotide according tothe invention. Such transformation vectors specific to the host organismto be transformed are well known to those skilled in the art and widelydescribed in the literature. In order to produce a polynucleotide orendolysin polypeptide according to the invention in a host, a processfor the transformation of a host organism, and integration of apolynucleotide, nucleic acid construct or expression vector according tothe invention may be appropriate. Such transformation may be carried outby any suitable known means which have been widely described in thespecialist literature and are well-known to the person skilled in theart.

In a third aspect, the invention relates to a host cell comprising apolynucleotide according to the invention, a nucleic acid constructaccording to the invention or an expression construct according to theinvention. A host cell according to the invention may be any microbial,prokaryotic or eukaryotic, cell which is suitable for expression of thepolypeptide of the invention. Preferably, said cell is an E. coli. In aneven more preferred embodiment, said cell is E. coli XL1blue MRF.

In a fourth aspect, the invention provides for a method for theproduction of an endolysin polypeptide according to the invention,comprising:

culturing a host cell according to the invention under conditionsconducive to the production of the endolysin polypeptide,

optionally isolating and purifying the endolysin polypeptide from theculture broth, and

optionally freeze-drying the endolysin polypeptide.

Preferably, an E. coli is used in the method for producing an endolysinpolypeptide according to the invention. More preferably an E. coliXL1blueMRF is used in step i) for producing an endolysin polypeptideaccording to the invention. Preferably, in the step of isolation andpurification, IMAC and Econo-Pac Chromatography columns (Biorad) packedwith 5 mL low density Nickel chelating agarose beads (ABT beads) incombination with gravity flow are used to purify an endolysinpolypeptide according to the invention. The eluted polypeptide can bedialyzed for 2, 4, and 12 hours against 3×11 lyophilization buffer, saidbuffer preferably comprising 50 mM phosphate, 500 mM sucrose, 200 mMmannitol, 0.005% polysorbate20, pH 7.4.

Lyophilisation and reconstitution are preferably construed asdehydration by freeze-drying and subsequent reconstitution of the sampleby adding water. Preferably, lyophilisation and reconstitution isperformed by dialyzing against 3 changes of 300 ml lyophilization buffer(50 mM phosphate or Tris, 500 mM sucrose, 200 mM mannitol, pH 7.4)aliquot and freezing in the gaseous phase of liquid nitrogen.Freeze-drying is preferably performed under standard conditions,preferably at −40° C. and vacuum at 75 mTorr for 60 minutes, followed byincreasing the temperature during 5 hours to −10° C. and another 60minutes at −10° C. at the same vacuum levels. As a final step, thetemperature is preferably increased to 25° C. during 10 hours. Samplesare reconstituted by the addition of water.

The invention also provides for a (claim 8) method for producing anendolysin polypeptide according to the invention with enhanced activitycomprising dialysis of an endolysin polypeptide according to the firstaspect of the invention, or an endolysin polypeptide obtainable by amethod depicted here above, said dialysis comprising the steps of:

-   -   i) dialysis against a buffer comprising a chelating compound,        and    -   ii) dialysis against a divalent metal ion-containing buffer,        preferably a divalent metal ion selected form the group        consisting of Co²⁺, Cu²⁺, Mg²⁺, Ca²⁺, Mn²⁺ and Zn²⁺.

A “chelating compound” is defined herein as a compound that binds ametal ion. Well known chelating compounds are ethylene diaminetetra-acetic acid (EDTA) and ethylene glycol tetra-acetic acid (EGTA).Preferably EDTA is used in step i) of the method of the invention.

Preferably, the divalent metal ion of step ii) is selected from thegroup consisting Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Cu²⁺, more preferably, saiddivalent metal ion is selected from the group consisting of Mg²⁺, Ca²⁺,Mn²⁺ and Co²⁺, even more preferably said divalent metal ion is Mg²⁺ orCa²⁺.

Substituting a divalent metal ion by any of Co²⁺, Cu²⁺, Mg²⁺, Ca²⁺, Mn²⁺and Zn²⁺ will preferably result in an increase of a lytic activity of anendolysin polypeptide according to the invention. Lytic activity isassessed as defined elsewhere herein. Preferably, this method accordingto the invention leads to an increase in a lytic activity of at least1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 fold as compared to thecorresponding untreated polypeptide. Even more preferably, the methodleads to an increase in lytic activity of at least 2.5 fold. Preferably,the treated polypeptide exhibits a 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 to 2 fold increase in lytic activity ascompared to the untreated polypeptide with the amino acid sequenceaccording to SEQ ID NO: 2.

In a fifth aspect, the invention provides for a composition comprisingan endolysin polypeptide according to the invention, or an endolysinpolypeptide obtainable by a method according to the invention, or apolynucleotide according to the invention, or a nucleic acid constructaccording to the invention, or an expression construct according to theinvention, or a host cell according to the invention. Such compositionaccording to the invention preferably exhibits lytic activity lytic forStaphylococcus, preferably said lytic activity is peptidoglycanhydrolase activity for Staphylococcus peptidoglycan. Preferably, saidStaphylococcus is selected from the group consisting of S. aureus, S.simulans and S. carnosus. A composition according to the presentinvention may comprise a mixture of different polynucleotides, and/ornucleic acid constructs and/or endolysin polypeptides an/or vectorsand/or cells according to the invention or obtainable by a methodaccording to the invention.

A composition according to the invention may further comprise apharmaceutically acceptable excipient. Such composition is hereinreferred to as a pharmaceutical composition and is preferably for use asa medicine or as a medicament. Preferably the medicament is used in thetreatment of infectious diseases.

Accordingly, there is provided a pharmaceutical composition comprisingan endolysin polypeptide according to the invention, or an endolysinpolypeptide obtainable by a method according to the invention, or apolynucleotide according to the invention, or a nucleic acid constructaccording to the invention, or an expression construct according to theinvention, or a host cell according to the invention, saidpharmaceutical composition further comprising a pharmaceuticallyacceptable excipient.

A composition according to the invention may further comprise one ormore additional active ingredients. Active preferably defined as showinga lytic activity as defined elsewhere herein. Preferably, said one ormore additional active ingredients are selected from the groupconsisting of a bacteriophage or phage, a phage endolysin derived fromsuch phage and an antibiotic. A phage encompassed herein can be anyphage known in literature. Preferably, such phage is, but is notlimited, from a family of the list consisting of Myoviridae,Siphoviridae and Podoviridae. Such phage may also be from a family ofthe list consisting of Tectiviridae, Corticoviridae, Lipothrixviridae,Plasmaviridae, Rudiviridae, Fuselloviridae, Inoviridae, Microviridae,Leviviridae and Cystoviridae. Within the context of the invention, acombination of active ingredients as defined herein can be administeredsequentially or simultaneously. A composition according to the inventionmay be in the liquid, solid or semi-liquid or semi-solid form.

A composition according to the invention can be used to treat animals,including humans, infected with Staphylococcus, preferably S. aureus.Any suitable route of administration can be used to administer saidcomposition including but not limited to: oral, aerosol or other devicefor delivery to the lungs, nasal spray, intravenous, intramuscular,intraperitoneal, intrathecal, vaginal, rectal, topical, lumbar puncture,intrathecal, and direct application to the brain and/or meninges.

A composition according to the invention comprising a polynucleotide ora nucleic acid construct or an endolysin polypeptide or a vector or acell according to the invention or obtainable by a method according tothe invention is preferably said to be active, functional ortherapeutically active or able to treat, prevent and/or delay aninfectious disease when it decreases the amount of a Staphylococcusspecies present in a patient or in a cell of said patient or in a cellline or in a cell free in vitro system and preferably means that 99%,90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the initialamount of a Staphylococcus species, is still detectable after treatment.Preferably no Staphylococcus species is detectable after treatment. Inthis paragraph, the expression “amount of Staphylococcus species”preferably means alive Staphylococcus genera. Staphylococcus species maybe detected using standard techniques known by the artisan such asimmunohistochemical techniques using Staphylococcus specific antibodies,tube coagulase tests that detect staphylocoagulase or “free coagulase”,detection of surface proteins such as clumping factor (slide coagulasetest) and/or protein A (commercial latex tests). Alive Staphylococcusspecies may be detected using standard techniques known by the artisansuch as microbiological bacterial culture techniques and/or real-timequantitative reverse transcription polymerase chain reaction to assayfor bacterial mRNA. Said decrease is preferably assessed in a tissue orin a cell of an individual or a patient by comparison to the amountpresent in said individual or patient before treatment with saidcomposition or polypeptide according to the invention. Alternatively,the comparison can be made with a tissue or cell of said individual orpatient which has not yet been treated with said composition orpolypeptide in case the treatment is local.

A composition comprising a polynucleotide or a nucleic acid construct ora polypeptide or a vector or a cell according to the invention orobtainable by a method according to the invention may be administered toa subject in need thereof or of a cell, tissue or organ or said patientat least one week, one month, six month, one year or more.

Accordingly, the invention provides for a composition according to theinvention, for use as a medicament for the treatment of a subject inneed thereof. Preferably, said composition is for use as a medicament inthe treatment of a condition associated with infection of a subject witha Staphylococcus, preferably a bacterium of the species S. aureus.

The invention further provides for a method of treatment, delay and/orprevention of a condition associated with infection of a subject with aStaphylococcus, preferably a bacterium of the species S. aureus,comprising administration an endolysin polypeptide according theinvention, or an endolysin polypeptide obtainable by a method accordingto the invention, or a polynucleotide according to the invention, or anucleic acid construct according to the invention, or an expressionconstruct according to the invention, or a host cell according to theinvention, or a composition according to the invention.

The medical use herein described may be formulated as a productaccording to the invention for use as a medicament for treatment of thestated diseases but could equally be formulated as a method of treatmentof the stated diseases using a product according to the invention, aproduct according to the invention for use in the preparation of amedicament to treat the stated diseases and use of a product accordingto the invention for the treatment of the stated diseases. Such medicaluses are all envisaged by the present invention. The subject in need oftreatment, delay and/or prevention of a condition associated withinfection may by any animal subject, preferably a mammal, morepreferably cattle, domestic animals like a dog or a cat, or a humansubject.

The endolysin polypeptide according the invention, or an endolysinpolypeptide obtainable by a method according to the invention, or apolynucleotide according to the invention, or a nucleic acid constructaccording to the invention, or an expression construct according to theinvention, or a host cell according to the invention, or a compositionaccording to the invention may conveniently be used in a method forcombination treatment of atopic dermatitis as presented in WO2015/005787wherein anti-inflammatory compound and a compound specifically targetinga bacterial cell, preferably a gram positive bacterial cell are used totreat atopic dermatitis or eczema. The compound specifically targeting abacterial cell can conveniently be an endolysin polypeptide obtainableby a method according to the invention, or a polynucleotide according tothe invention, or a nucleic acid construct according to the invention,or an expression construct according to the invention, or a host cellaccording to the invention, or a composition according to the invention.

The endolysin polypeptide according the invention, or an endolysinpolypeptide obtainable by a method according to the invention, or apolynucleotide according to the invention, or a nucleic acid constructaccording to the invention, or an expression construct according to theinvention, or a host cell according to the invention, or a compositionaccording to the invention may conveniently be used in a method oftreatment of an intracellular bacterial infection in a subject in needthereof, comprising:

-   -   administration of an effective amount of an agent that increases        the intracellular pH of a host cell and/or of an intracellular        compartment of a host cell, and    -   administration of an effective amount of a bactericidal agent.        Said method is herein referred to as a method according to the        invention,        wherein preferably, the increase in pH activates a        non-replicating intracellular bacterium and the bactericidal        agent kills the activated intracellular bacterium. Such method        of treatment is described in EP15158880.3. The bactericidal        agent can conveniently be an endolysin polypeptide obtainable by        a method according to the invention, or a polynucleotide        according to the invention, or a nucleic acid construct        according to the invention, or an expression construct according        to the invention, or a host cell according to the invention, or        a composition according to the invention. Preferably, the        endolysin polypeptide according to the invention comprises a        protein transduction domain enabling the endolysin polypeptide        to enter the cell that harbors the intracellular bacterium.        Protein transduction domains are extensively described in        EP15158880.3.

In an aspect, the invention relates to a non-medical compositionexhibiting lytic activity as defined elsewhere herein. Preferably, thisembodiment relates to an antimicrobial. Preferably, this embodimentrelates to an antimicrobial for lysing a bacterium, preferably abacterium of the genus Staphylococcus, more preferably a bacterium ofthe species S. aureus. Preferably this embodiment relates to anantimicrobial as food preservative or disinfectant.

Accordingly, there is provided the use of an endolysin polypeptideaccording to the invention, or an endolysin polypeptide obtainable by amethod according to the invention, or a polynucleotide according to theinvention, or a nucleic acid construct according to the invention, or anexpression construct according to the invention, or a host cellaccording to the invention, or a composition according to the invention,as an antimicrobial, preferably as a food additive or a disinfectant.Examples of such use are, but are not limited to, rinsing the cups of amilking device with a composition according to the invention beforemilking to prevent transmission of Staphylococci from cow to cow,cleaning of surfaces in food industry and cleaning chirurgical tools.Such use can be combined with any sterilization method or disinfectantknown in the art such as ultrasonic cleaning, irradiation or thermalsterilization, by immersing the equipment in a disinfectant solutionsuch as ethanol, ammonium, iodine and/or aldehyde disinfectant, or byusing gas sterilization by retaining the device in a closed atmospheresuch as formalin gas or ethylene oxide gas.

The invention further relates to an endolysin polypeptide according tothe invention, or an endolysin polypeptide obtainable by a methodaccording to the invention, or a polynucleotide according to theinvention, or a nucleic acid construct according to the invention, or anexpression construct according to the invention, or a host cellaccording to the invention, or a composition according to the invention,for detecting a Staphylococcus, preferably S. aureus, preferably in adiagnostic application.

Definitions

“Sequence identity” is herein defined as a relationship between two ormore amino acid (peptide, polypeptide, or protein) sequences or two ormore nucleic acid (nucleotide, polynucleotide) sequences, as determinedby comparing the sequences. In the art, “identity” also means the degreeof sequence relatedness between amino acid or nucleotide sequences, asthe case may be, as determined by the match between strings of suchsequences. “Similarity” between two amino acid sequences is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one peptide or polypeptide to the sequence of a secondpeptide or polypeptide. In a preferred embodiment, identity orsimilarity is calculated over the whole SEQ ID NO as identified herein.“Identity” and “similarity” can be readily calculated by known methods,including but not limited to those described in Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heine, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux,J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP,BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410(1990). The BLAST X program is publicly available from NCBI and othersources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md.20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and GapLength Penalty: 4. A program useful with these parameters is publiclyavailable as the “Ogap” program from Genetics Computer Group, located inMadison, Wis. The aforementioned parameters are the default parametersfor amino acid comparisons (along with no penalty for end gaps).Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to ser; Arg to lys; Asn to gln or his; Asp toglu; Cys to ser or ala; Gln to asn; Glu to asp; Gly to pro; His to asnor gln; Ile to leu or val; Leu to ile or val; Lys to arg; gln or glu;Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trpto tyr; Tyr to trp or phe; and, Val to ile or leu.

A “nucleic acid molecule” or “polynucleotide” (the terms are usedinterchangeably herein) is represented by a nucleotide sequence. A“polypeptide” is represented by an amino acid sequence. A “nucleic acidconstruct” is defined as a nucleic acid molecule which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acids which are combined or juxtaposed in a mannerwhich would not otherwise exist in nature. A nucleic acid molecule isrepresented by a nucleotide sequence. Optionally, a nucleotide sequencepresent in a nucleic acid construct is operably linked to one or morecontrol sequences, which direct the production or expression of saidpeptide or polypeptide in a cell or in a subject.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed at a position relative to thenucleotide sequence coding for the polypeptide of the invention suchthat the control sequence directs the production/expression of thepeptide or polypeptide of the invention in a cell and/or in a subject.“Operably linked” may also be used for defining a configuration in whicha sequence is appropriately placed at a position relative to anothersequence coding for a functional domain such that a chimeric polypeptideis encoded in a cell and/or in a subject.

“Expression” is construed as to include any step involved in theproduction of the peptide or polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification and secretion.

A “control sequence” is defined herein to include all components whichare necessary or advantageous for the expression of a polypeptide. At aminimum, the control sequences include a promoter and transcriptionaland translational stop signals. Optionally, a promoter represented by anucleotide sequence present in a nucleic acid construct is operablylinked to another nucleotide sequence encoding a peptide or polypeptideas identified herein.

The term “transformation” refers to a permanent or transient geneticchange induced in a cell following the incorporation of new DNA (i.e.DNA exogenous to the cell). When the cell is a bacterial cell, as isintended in the present invention, the term usually refers to anextrachromosomal, self-replicating vector which harbors a selectableantibiotic resistance.

An “expression vector” may be any vector which can be convenientlysubjected to recombinant DNA procedures and can bring about theexpression of a nucleotide sequence encoding a polypeptide of theinvention in a cell and/or in a subject. As used herein, the term“promoter” refers to a nucleic acid fragment that functions to controlthe transcription of one or more genes or nucleic acids, locatedupstream with respect to the direction of transcription of thetranscription initiation site of the gene. It is related to the bindingsite identified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites, and any other DNA sequences,including, but not limited to, transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter.Within the context of the invention, a promoter preferably ends atnucleotide −1 of the transcription start site (TSS).

A “polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules. The sequenceinformation as provided herein should not be so narrowly construed as torequire inclusion of erroneously identified bases. The skilled person iscapable of identifying such erroneously identified bases and knows howto correct for such errors.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a product or a composition or anucleic acid molecule or a peptide or polypeptide of a nucleic acidconstruct or vector or cell as defined herein may comprise additionalcomponent(s) than the ones specifically identified; said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the elements ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

FIGURE LEGENDS

FIG. 1. Representative image of minimal inhibitory concentrations (MIC)of an endolysin polypeptide according to the invention.

FIG. 2A and FIG. 2B. Thermal stability and specific activity ofendolysin polypeptide according to the invention.

FIG. 3. Turbidity reduction assays comparing SA.100 (upper pane and midpane) with XZ.700 (lower pane).

EXAMPLES Materials and Methods

Bacterial Strains, Culture Conditions, Phages and Plasmids

E. coli XL1BlueMRF and E. coli Sure was used for the over-expression ofpolypeptides according to the invention, essentially as inWO2013/169102. Both strains were cultured in LB-PE medium at 30° C. with100 μg/ml ampicillin and 30 μg/ml tetracycline for plasmid selection.

DNA Techniques and Cloning Procedures

Standard techniques according to Sambrook, Maniatis et al. (1989) wereemployed for cloning.

Expression and Purification of Recombinant Endolysin Polypeptides

Protein overexpression and partial purification was essentially done aspreviously described by others (Loessner et al., 1996, Schmelcher etal., 2010). In brief, plasmid bearing E. coli were grown in 250 mlmodified LB medium (15 g/l tryptose, 8 g/l yeast extract, 5 g/l NaCl, pH7.8) to an optical density at 600 nm (OD600 nm) of 0.4 to 0.6 andinduced with 1 mM IPTG. Cells were further incubated for 4 hours at 30°C., or 18 hours at 20° C., cooled to 4° C., and harvested bycentrifugation. Cell pellets were suspended in 5 ml immobilizationbuffer (50 mM NaH₂PO₄, 500 mM NaCl, 5 mM imidazole, 0.1% polysorbate20,pH 7.4). Cytosolic E. coli contents containing soluble recombinantproteins were liberated by a double passage through a French PressureCell Press (1200 psi, SLM Aminco, Urbana, Ill., U.S.) operated at 1200psi. Other downstream processing steps included removal of insolublecell debris by centrifugation, filter sterilization (0.2 μm PESmembrane, Millipore), and Immobilized Metal Affinity Chromatography(IMAC) purification using MicroBiospin (Bio-Rad, Hercules, Calif., U.S.)columns packet with low density Ni-NTA Superflow resin (ChemieBrunschwig AG, Basel, Switzerland). Ni-NTA immobilized proteins wereon-column gravity flow washed with 5-10 column volumes immobilizationbuffer. Protein fractions were then eluted with elution buffer (50 mMNaH₂PO₄, 500 mM NaCl, 125 mM imidazole, 0.1% polysorbate20, pH 7.4) anddialyzed against two changes of dialysis buffer (50 mM NaH₂PO₄, 100 mMNaCl, 0.1% polysorbate20, pH 7.4). Protein concentrations were definedin a NanoDrop ND-1000 spectrophotmeter, corrected for specificabsorbance at 280 nm as calculated from the primary amino acid sequencewith Vector NTI software (Invitrogen, Carlsbad, Calif., U.S.) andestimated for purity by SDS-PAGE. Aliquots were stored at −20° C. mixedwith 50% glycerol.

Lyophilization of Recombinant Proteins

IMAC purified proteins were dialyzed against 3 changes of 300 mllyophilization buffer (50 mM phosphate or Tris, 500 mM sucrose, 200 mMmannitol, pH 7.4) aliquot and frozen in the gaseous phase of liquidnitrogen. The freeze-drying was done at −40° C. and vacuum at 75 mTorrfor 60 minutes, followed by increasing temperature during 5 hours to−10° C. and another 60 minutes at −10° C. at the same vacuum levels. Asfinal step, temperature was increased to 25° C. during 10 hours. Sampleswere reconstituted prior to testing in lysis assays by the addition ofwater.

Turbidity Lysis Assays

Substrate cells for lytic activity assays were grown to an opticaldensity at 595 nm (OD595) of 0.4, washed twice with PBST pH 7.4 andre-suspended in 15% glycerol containing PBS buffer pH 7.4 concentratingit at the same time 100 fold. The cells were stored at −20° C. Forfurther use in binding or lytic activity assays the cells were thaw,washed with PBS pH 7.4 and diluted to an OD595 of 1±0.05. In standardlytic activity assays protein samples were diluted to equimolar amountsand distributed in transparent 96-well tissue culture test plates (SPLlife sciences, Pocheon, Korea). Substrate cells were added to a finalvolume and drop in optical density at 595 nm (OD595 nm) were recordedfor about 1 hour at 37° C.

Decrease in optical density at 595 nm (OD595) was measured using aVictor3 1420 Multilabel Counter instrument (Perkin Elmer) during 1 hour.Plates were shaken vigorously for 1 second (double orbit, 0.1 mmdiameter) after every single read out. As positive control servedN-terminal 6× His tagged Lysostaphin (HLST), commercially availableLysostaphin (recombinant, E. coli originated, Sigma). As negativecontrol we applied MilliQ water.

Minimal Inhibitory Concentration (MIC) Assay

Two-fold serial dilutions (2000 nM to 4 nM) of endolysin in caseinpeptone soybean flour peptone broth (CASO broth (Carl Roth, Germany))were distributed in sterile crystal clear 96-well plates at 100 μl/well.S. aureus Newbold were grown in CASO broth to mid log phase (OD600 nm˜0.5) and diluted with fresh CASO broth to ˜1E+6 cfu/ml. The cells werethen distributed in the endolysin containing wells resulting inendolysin concentrations of 1000 nM to 2 nM and ˜10E+5 cfu/well. The96-well plates were covered with the lid and incubated for 20 hours at30° C. Images were takes using a gel scanner. As positive control servedCASO broth without endolysin, and as negative control CASO broth withoutS. aureus cells was used.

Results

Determination of Specific Activity of an Endolysin Polypeptide Accordingto the Invention by Minimal Inhibitory Concentration Analysis

The specific activity of XZ.700, an endolysin polypeptide with an aminoacid sequence according to SEQ ID NO: 2 was compared to that of SA.100,a reference endolysin polypeptide with an amino acid sequence accordingto SEQ ID NO: 1. The results are depicted in FIG. 1. MIC values wereobtained by exposure of 2-3E+5 cfu S. aureus Newbold to serial dilutionsof Staphefekt in a 200 μl format. The image was taken after 20 hincubation in CASO medium. The MIC value of SA.100 was 62.5 nM and MICvalue of XZ.700 was 15.6 nM in three independent assays; i.e. the MICvalue of XZ.700 is four-fold lower that of SA.100, indicative of afour-fold higher specific activity of XZ.700.

Determination of Thermal Stability and Specific Activity of an EndolysinPolypeptide According to the Invention by Turbidity Assay

The thermal stability and specific activity of XZ.700, an endolysinpolypeptide with an amino acid sequence according to SEQ ID NO: 2 werecompared to those of SA.100, a reference endolysin polypeptide with anamino acid sequence according to SEQ ID NO: 1. In brief, 200 mM ofSA.100 and XZ.700 were subjected to 10 minutes heat exposure (X-axis)followed by cooling on ice. The relative activity in view of SA.100 at20 degrees Celsius was subsequently determined by turbidity lysis assay.It can clearly be observed in FIG. 2 that the activity of XZ.700 issomewhat lower than SA.100 from about 55 degrees Celsius and higher,whereas the activity of XZ.700 is significantly higher than SA.100 up toabout 55 degrees Celsius. At room temperature, the activity of ZX.700 isabout 70% higher than SA.100.

Specific activity and thermal stability were also determined using akinetic assay. XZ.700 and SA.100 at concentrations of 200 nM were heatexposed between 42 degrees Celsius and 72 degrees Celsius for 10 minutesin PBS buffer in a gradient thermocycler. After subsequent cooling onice, lysis kinetics in standard dynamic turbidity reduction assays wererecorded at 100 nM protein concentrations against S. aureus SA.113 inPBS buffer pH 7.4. The lysis curves are depicted in FIG. 3 and showoptical density reductions (Y-axis, initial OD600 nm ˜1) over time(X-axis, duration 1 h). It can clearly be observed that up to 60 degreesCelsius ZX.700 has a significantly steeper curve than SA.100, indicatinghigher specific activity.

Immunogenicity Prediction of an Endolysin Polypeptide According to theInvention.

Immunogenicity predictions were performed on the XZ.700 and SA.100polypeptide sequences (SEQ ID NO: 1 and SEQ ID NO: 2, respectively)using the tools.immuneepitope.org/bcell/webpage with parameters: centreposition 4, window size 7, and threshold 1.0. The results are depictedin Table 1. It is clear that the immunogenic peptide fragment“VKELKHIYSNH” (position 197-207 in SA.100) is not present in XZ.700.

Altogether, in this non-limiting example, it was demonstrated thatendolysin polypeptide according to the invention XZ.700 has a higherspecific activity and lower immunogenicity than prior art endolysinpolypeptide SA.100.

TABLE 1Kolaskar & Tongaonkar Immunogenicity prediction of peptides present in Staphefekt SA.100 (left) and Staphefekt XZ.700 (right).

SEQUENCES

SEQ ID NO: 1 MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGELLRPKDAKKDEKSQVCSGLAMEKYDITNLNAKQDKSKNGSVKELKHIYSNHIKGNKITAPKPSIQGVVIHNDYGSMTPSQYLPWLYARENNGTHVNGWASVYANRNEVLWYHPTDYVEWHCGNQWANANLIGFEVCESYPGRISDKLFLENEEATLKVAADVMKSYGLPVNRNTVRLHNEFFGTSCPHRSWDLHVGKGEPYTTTNINKMKDYFIKRIKHYYDGGKLEVSKAATIKQSDVKQEVKKQEAKQIVKATDWKQNKDGIWYKAEHASFTVTAPEGIITRYKGPWTGHPQAGVLQKGQTIKYDEVQKFDGHVWVSWETFEGETVYMPVRTWDAKT GKVGKLWGEIK SEQ ID NO: 2MAATHEHSAQWLNNYKKGYGYGPYPLGINGGMHYGVDFFMNIGTPVKAISSGKIVEAGWSNYGGGNQIGLIENDGVHRQWYMHLSKYNVKVGDYVKAGQIIGWSGSTGYSTAPHLHFQRMVNSFSNSTAQDPMPFLKSAGYGKAGGTVTPTPNTGLKHIYSNHIKGNKITAPKPSIQGVVIHNDYGSMTPSQYLPWLYARENNGTHVNGWASVYANRNEVLWYHPTDYVEWHCGNQWANANLIGFEVCESYPGRISDKLFLENEEATLKVAADVMKSYGLPVNRNTVRLHNEFFGTSCPHRSWDLHVGKGEPYTTTNINKMKDYFIKRIKHYYDGGKLEVSKAATIKQSDVKQEVKKQEAKQIVKATDWKQNKDGIWYKAEHASFTVTAPEGIITRYKGPWTGHPQAGVLQKGQTIKYDEVQKFDGHVWVSWETFEGETVYMPVRTWDAK TGKVGKLWGEIK

The invention claimed is:
 1. A method of treatment of atopic dermatitisor eczema comprising administrating to a subject in need thereof anendolysin polypeptide specific for Staphylococcus, said polypeptidecomprising: a polypeptide with an amino acid sequence that has at least90% sequence identity with SEQ ID NO: 2 over its entire length, or apolypeptide with an amino acid sequence that has at least 90% sequenceidentity with SEQ ID NO: 1 and having a truncation of at least 5, 10,20, 30, or 40 amino acids in the region of amino acids 156 to 199 of SEQID NO: 1, wherein the endolysin polypeptide has enhanced specificactivity and/or reduced immunogenicity than an endolysin polypeptidewith the amino acid sequence according to SEQ ID NO: 1, and wherein thepolypeptide is a synthetic molecule.
 2. The method of treatmentaccording to claim 1, wherein the endolysin polypeptide comprises theamino acid sequence according to SEQ ID NO:
 2. 3. A method of treatmentaccording to claim 1, wherein the endolysin polypeptide is administeredwithin a composition.
 4. The method of treatment according to claim 3,wherein the composition further comprises a pharmaceutically acceptableexcipient.
 5. The method of treatment according to claim 3, wherein thecomposition further comprises an additional active ingredient.
 6. Themethod of treatment according to claim 5, wherein the additional activeingredient is selected from the group consisting of a bacteriophage orphage, a phage endolysin derived from such phage and an antibiotic. 7.The method of treatment according to claim 3, wherein the composition isin liquid, solid, semi-liquid or semi-solid form.
 8. The method oftreatment according to claim 3, wherein the subject is an animalsubject.
 9. The method of treatment according to claim 8, wherein thesubject is cattle or a domestic animal, or wherein the subject is ahuman.